1. Field of the Invention
The present invention relates to a method for measuring characteristic micromotions created by chemical, mechanical, optical, or electrical processes using scanning probe microscopy or laser interferometry. In particular, a stationary mode atomic force microscope is used to monitor microscopic dynamic processes, such as the replication of DNA for DNA sequencing.
2. Description of Related Art
The development of scanning probe microscopes in the 1980s, such as the scanning tunneling microscope and the atomic force microscope, provided the opportunity to locate and identify microscopic sites with atomic resolution. A wide variety of sites can be observed: small biological molecules that perform sophisticated biological functions, atomic sites on the surface of materials where corrosion, gasification, or catalytic reactions take place, and atomic sites where stress-induced fracture occurs.
The scanning tunneling microscope (STM) has a fine conducting probe that is held close to the surface of a site. Electrons tunnel between the site and the probe, producing an electrical signal. The probe is moved slowly across the surface and raised and lowered so as to keep the signal constant. A profile of the surface is produced, and a computer-generated contour map of the surface is generated. The technique is capable of resolving individual atoms, but requires conductive materials for image formation.
The atomic force microscope (AFM) also has a small probe that is held on a spring-loaded or flexible cantilever in contact with the surface of a site. The probe is moved slowly across the surface, and the tracking force between the tip and the surface is monitored. Forces as small as 10.sup.-13 N can be measured. The probe is raised and lowered so as to keep this force constant, and a profile of the surface is produced. Typically, a laser beam is bounced off the cantilever to monitor its position with angstrom-scale precision. Scanning the probe over the site at a constant force gives a computer-generated contour map of the surface. This instrument is similar to the STM, but uses mechanical forces rather than electrical signals. The AFM can resolve individual molecules and, unlike the STM, can be used with non-conducting samples, such as biological specimens.
A number of research groups, including ones at Lawrence Livermore National Laboratory, have attempted, with limited success, to use the scanning probe microscopes to sequence DNA by resolving individual nucleotide bases in tunneling images. Sequencing the human genome is one of the major scientific goals in the United States and in the world today. Dramatic improvements in human health and well-being will be possible by understanding the ordering of the billions of base pairs contained in DNA. Major research projects, like the Human Genome Project, have a need for improved techniques that will significantly reduce the analysis time of DNA fragments. The demands on the sequencing technology are even greater than the Genome Project because scientists also want to understand the DNA of different animals, plants, and microorganisms as quickly as possible.
Presently, the most common approach for sequencing the human genome is electrophoresis, which uses an electric field to separate fragments of DNA as they migrate through a sieving matrix (a gel). The DNA fragments are produced in a number of ways. The most widely employed method uses restriction enzymes, which act as molecular scissors, to sever the DNA at precise locations, producing a unique family of fragments for each enzyme used. The necessary number of DNA fragments are produced by biochemical reactions such as the DNA polymerase chain reaction (PCR), which can make millions of copies of a given DNA sequence. Unfortunately, these conventional electrophoresis techniques are relatively slow and costly, and time estimates for mapping or sequencing the entire human DNA molecule using these techniques range from decades to centuries.
Likewise, the use of STM and AFM in the conventional scanning or visualizing mode to sequence DNA presents problems. The normal process of raster scanning the microscope tip across the DNA molecules causes the molecules to move. In addition, the poor conductivity of the DNA precludes STM observation of the bases as attached to the phosphate backbone. The difficulty with the conventional AFM approach is that the radius of curvature of even the best AFM tips interferes with the identification of DNA bases.
U.S. Pat. No. 5,106,729 by Lindsay et al. describes a method for determining and visualizing the base sequence of DNA and RNA with a scanning probe microscope. The method replaces the oxygen in the DNA with a metal-sulfur complex, and passes the probe over the complexed polymer to measure and record the differences in electrical conductivity at preselected increments along the scanning path. Lindsay acknowledges the limitations of STM and AFM, caused by the distortion of the soft molecules as the tip touches them. Lindsay attempts to solve this problem by using the metal complex to enhance the electrical contrast in the STM.
However, an urgent need exists for a DNA sequencing technique that is faster than the conventional electrophoresis techniques, and that does not require biochemical labeling or complexing the DNA molecule in order to image the base sequence. This invention addresses these challenges in particular, and introduces a general technique for detecting and measuring micromotions caused by an unlimited variety of chemical, mechanical, optical, and electrical processes.